Composition for promoting neuroregeneration, containing extracellular vesicles derived from three-dimensional spheroidal cell aggregates

Abstract

present invention relates to: a composition for promoting neurogenesis, containing extracellular vesicles prepared by a novel preparation method and derived from three-dimensional spheroid-type cell aggregate; and a neurogenesis promotion method. Extracellular vesicles prepared by a novel preparation method, of the present invention, can improve neuroplasticity by inducing neurogenesis, neuronal differentiation or neural circuit restoration, and thus can be effectively used in the treatment of various diseases requiring neurogenesis.

1 . A method for preventing or treating neurological diseases and damage comprising;
 (a) preparing a three-dimensional spheroid-type cell aggregate by 3-dimensionally (3D) culturing stem cells in a microwell with a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and   (b) preparing extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate by isolating extracellular vesicles from the three-dimensional spheroid-type cell aggregate; and   (c) treating the extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate prepared in step (b) to a subject in need thereof.   
     
     
         2 . The method of  claim 1 , wherein the stem cells are at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells and embryonic stem cells. 
     
     
         3 . The method of  claim 1 , wherein the 3D culture in step (a) is static culture. 
     
     
         4 . The method of  claim 1 , wherein the 3D culture in step (a) is performed by dispersing and culturing the mesenchymal stem cells in a microwell at a density of 200 to 600 cells/well. 
     
     
         5 . The method of  claim 1 , wherein the extracellular vesicles highly express at least one selected from the group consisting of miR-27a, miR-132, miR-146a, and miR-146b as compared with extracellular vesicles derived from spheroidal cell aggregates obtained by 3D dynamic culture of mesenchymal stem cells and extracellular vesicles derived from mesenchymal stem cells obtained by 2D culture. 
     
     
         6 . The method of  claim 1 , wherein the extracellular vesicles highly express at least one selected from the group consisting of vascular endothelial growth factor (VEGF), a brain-derived neurotrophic factor (BDNF), a brain-derived neurotrophic factor (FGF) and a brain-derived neurotrophic factor (NGF) as compared with extracellular vesicles derived from spheroidal cell aggregates obtained by 3D dynamic culture of mesenchymal stem cells and extracellular vesicles derived from mesenchymal stem cells obtained by 2D culture. 
     
     
         7 . The method of  claim 1 , wherein the neurological diseases and damage are at least one disease and damage selected from the group consisting of spinal cord injury, Parkinson's disease, stroke, amyotrophic spinal lateral sclerosis, motor nerve damage, peripheral nerve damage due to trauma, nerve damage due to ischemic brain damage, neonatal hypoxic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease, congenital metabolic nervous system disease, and traumatic brain injury. 
     
     
         8 . The method of  claim 1 , wherein the composition is used for inducing neurogenesis, neuronal differentiation, or neural circuit restoration. 
     
     
         9 . The method of  claim 8 , wherein the neural circuit restoration induction is identified in an MRI diffusion-tensor image (DTI). 
     
     
         10 . A method of  claim 1 ,
 wherein the extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate are contained in food composition or pharmaceutical composition.   
     
     
         11 . An in vitro method for promoting neurogenesis comprising:
 (a) preparing three-dimensional spheroid-type cell aggregate by 3-dimensionally (3D) culturing stem cells in a microwell with a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and   (b) isolating extracellular vesicles from the three-dimensional spheroid-type cell aggregate; and   (c) treating the extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate prepared in step (b) to in vitro cells   
     
     
         12 . A method for producing a composition for promoting neurogenesis comprising extracellular vesicles derived from three-dimensional spheroid-type cell aggregate, comprising:
 (a) preparing three-dimensional spheroid-type cell aggregate by 3-dimensionally (3D) culturing stem cells in a microwell with a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and   (b) isolating extracellular vesicles from the three-dimensional spheroid-type cell aggregate.   
     
     
         13 . (canceled)